1. Field of the Invention
This invention relates to cell staining. Specifically this invention relates to staining a Pap slide specimen, including conventional smears and ThinPrep Pap test slides.
2. Background and Discussion of the Prior Art
Almost 5,000 women die each year from cervical cancer in the United States. The cervico-vaginal Papanicolaou test or Pap test is a powerful tool for detecting cancerous and precancerous cervical lesions. The Pap test smear has been credited with reducing mortality from cervical cancer by as much as 70%.
The Pap test involves a staining method. This staining method includes a polychromatic reaction which seeks to display the many variations of cellular morphology to show degrees of cellular maturity and metabolic activity. The four main steps of the Pap test staining method are: (1) slide fixation, (2) nuclear staining with hematoxylin, (3) cytoplasmic staining, generally with counterstains orange G and EA, and (4) clearing and mounting. The term "Pap staining method" or "Pap test staining method" as used hereinbefore and hereinafter throughout the specification refers generally to the aforesaid method.
The Pap test staining method has been generally used on conventional cervico-vaginal Pap smear specimens. The conventional Pap test smear however had false negative rates ranging from 10-15%, and up to 90% of those false negative rates were due to limitations of staining and slide preparation of such specimens. More recently the Cytyc Corporation developed the ThinPrep Pap test. Instead of smearing the cells on a slide, the cells are collected in a transport medium, from which a slide with a filter preparation is obtained for the test.
The art provided several complex Pap test staining methods involving multiple steps of greatly varying times and clippings. Certain prior art short versions of Pap test staining methods took an inordinate amount of time of upwards to 20 minutes or more and were complex, which readily invited error by the laboratory technician. Many of these prior art Pap test staining short methods were generally directed to fine needle aspirated (FNA) specimens and to the conventional cervico-vaginal smears, and not to the ThinPrep Pap test specimen.
One such conventional prior art short method which sought to reduce the overall time, was the "Quick Papanicolaou Staining Procedure for Stat Specimens" developed by the Johns Hopkins Cytopathology Laboratory, Baltimore, Md., as follows:
1. Tap water 5-10 dips (until surface is smooth) 2. Gill's hematoxylin No. 2 1 min, including 10 initial dips 3. Tap water 5 dips 4. Scott's tap water substitute 15 sec 5. Tap water 5 dips 6. Stat OG/EA 1 min, including 10 initial dips 7. 95% ethanol 5 dips 8. 95% ethanol 5 dips 9. Absolute ethanol 10 dips 10. Absolute ethanol 10 dips 11. Xylene 5 dips 12. Xylene 5 dips Coverslip
The Johns Hopkins' Method required 12 post-fixing, pre-coverslip steps, and took several minutes. The great disparity in procedure times for each step and sequence invited error by the laboratory technician and difficult the mechanization of the staining method.
Another attempt to reduce the Pap test staining method time was done specifically in connection with fine needle aspiration (FNA) specimens as disclosed in "Ultrafast Papanicolaou Stain--An Alternative Preparation for Fine Needle Aspiration Cytology" G. C. H. Yang and I. I. Alvarez, ACTA Cytol., vol. 39, no. 1, January-February 1995. The Yang-Alvarez FNA staining method was as follows:
 A. Alcoholic Formalin Fixative: Mixture of 65% ethanol and 4% formaldehyde. It was convenient to make 3L of fixative from 300 mL of 38-40% formaldehyde, 2,053 mL of 95% ethanol and 647 mL of distilled water. B. Staining Method: 1. Normal saline 30 seconds 2. 95% Ethanol (optional, for storage/transport) 3. Alcoholic formalin 10 seconds 4. Water 6 slow dips 5. Richard-Allan Hematoxylin 2 2 slow dips 6. Water 6 slow dips 7. 95% Ethanol 6 slow dips 8. Richard-Allan Cytostain 4 slow dips 9. 95% Ethanol 6 slow dips 10. 100% Ethanol 6 slow dips 11. Xylene 10 slow dips Mount and coverslip
The Yang-Alvarez FNA staining method required a relatively complicated fixative mixture solution, 8 post-fixing, pre-coverslip steps, and provided improvement in the resultant stained specimen, but again the laboratory technician was faced with greatly disparate dipping sequences and times, and as such invited error. That is, the laboratory technician had to change from a 10 second fixing to a 6 dip hydrating and then to a 2 dip hematoxylin staining. These disparate sequencing times invited error. Further the Yang-Alvarez staining method was FNA specimen specific and not for cervico vaginal specimens.
One attempt was made to apply the Yang-Alvarez FNA approach to cervicovaginal smears as disclosed in "Ultrafast Papanicolaou Protocol for Cervicovaginal Smears," G. C. H. Yang et al., ASC Abstracts, Nov. 1, 1995. This modification of the Yang-Alvarez FNA staining method to cervicovaginal smears is as follows:
 1. Dist. water 3 min 2. Dist. water 3 min 3. Dist. water 3 min 4. Hexatox II 20 sec 5. Tap water until clear (discard dirty water fill with clean water) 6. 95% alcohol 6 dips 7. Cytostain 40 sec-5 min 8. 95% alcohol until clear (discard dirty alcohol, fill with clean alcohol) 9. 100% alcohol 6 dips 10. 100% alcohol 6 dips 11. Xylene 6 dips 12. Xylene 6 dips
The Yang et al adaption or modification of the Yang-Alvarez FNA staining method required 12 separate post-fixing, pre-coverslip steps, and also required more than 15 minutes, all in disparate sequence times ranging from a 6 dip step taking about 6 seconds to a cytostain staining step taking upwards of 5 minutes. Again this method was not laboratory technician friendly in that it invited errors because of the disparate dip sequencing as well as in the number of steps and in the disparate step times, and difficult the use of automatic stainer machines.
The art desired a simple, faster and laboratory technician friendly Pap test staining method. The art also desired a Pap test staining method used for conventional pap smears and which was particularly useful for ThinPrep Pap test specimens. The art further desired a Pap test staining method as aforesaid which consequently is adaptable for handling large numbers of Pap test slide specimens with minimal or no errors, and will facilitate the use of automatization for the staining method. The art still further required a method as aforesaid with an improved stained specimen, particularly an improvement in background and contrast stain quality. The present invention addresses and provides those art desired improvements.